| Objective:Through the establishment of extraction and ultrahigh performance liquid chromatoc-tandem mass spectrometry(UPLC-MS/MS)detection methods of saxitoxin and its metabolites,which are dicarbamoyl-saxitoxin and neosaxitoxin in biological materials,this paper provides a basis for the detection of poisons in actual poisoning cases.To achieve rapid qualitative and quantitative detection of saxitoxin,dicarbamoylsaxitoxin and neosaxitoxin in blood,urine,liver and other biological tissues.Through the establishment of acute poisoning rat model,the concentration changes of saxitoxin and its metabolites in rats with different administration methods and dosages were studied,which provided the basis for further study of toxicokinetics.By studying the distribution of saxitoxin and its metabolites in poisoned dead rats,the concentration data of poison in each tissue were obtained,which provided the basis for the selection of materials for poisoning(death)cases.Methods:1.Extraction and detection methodsBlood samples to be tested were taken 0.2m L,added with acetonitrile-0.1% acetic acid methanol(1:3,v/v).After shock and centrifugation,supernatant was taken for membrane detection.Urine sample 0.2m L was extracted with methanol(containing 0.5%acetic acid),the supernatant was taken after shaking and centrifugation,added to an activated PA solid phase extraction column,eluted with methanol-water(2:8,v:v)elution solution containing 0.5% acetic acid,and the eluate was fed into the sample after passing through the membrane.Liver tissue(grinded)was added to dilute phosphate buffer solution,shaken,centrifuged,and the supernatant was added to an activated Oasis WCX solid phase extraction column,eluted with methanol(containing 4% formic acid)with deionized water and acetonitrile,and the eluate was fed into the sample after passing through the membrane.The retention time and relative abundance ratio of ion pairs were qualitatively determined by UPLC-MS/MS method and quantified by external standard curve method.2.Animal experiments2.1 The experimental animals were healthy male rats provided by SPF(Beijing)Biotechnology Co.,Ltd.2.2 Acute saxitoxin poisoning rat model:2.2.1 Animal model of oral gavage: Low dose group: Twelve healthy male rats were randomly divided into 4 groups: 3 in blank group,3 in each of 6μg/kg,9μg/kg and18μg/kg groups.Adaptive feeding for one week,fasting the day before the experiment.Oral intragastric administration,blank group was given the same amount of normal saline to observe the poisoning symptoms.After administration,0.2m L blood was collected from the inner canthus venous plexus at 5min,10 min,30min,1h,2h,3h,4h,5h,6h,and 7h.The rats were killed 7h later,and urine was collected during the experiment and stored at-20℃ for examination.High-dose group: Twelve healthy male rats were randomly divided into 4 groups: 3 in blank group,3 in each of 36μg/kg,72μg/kg and108μg/kg groups,and the other operations were the same as above.2.2.2 Animal model by caudal vein injection: Eighteen healthy male rats were selected and randomly divided into 12μg/kg,9μg/kg,6μg/kg,4μg/kg,3μg/kg dose administration group and blank group.The rats were fed adaptively for one week and fasted the day before the experiment.The rats were injected into the tail vein,and the blank group was given the same amount of normal saline to observe the poisoning symptoms.After administration,0.2m L blood was collected from the inner canthus venous plexus at 1min,5min,10 min,30min,1h,1.5h,2h,2.5h,3h,4h,5h and 6h.The rats were killed 6h later,and blood at the time of death and urine were collected during the experiment and stored at-20℃ for examination.2.3 Postmortem distribution modelTwelve healthy male rats were fed for one week,fasted overnight before the experiment,6 were used as blank control,the rest were given 12μg/kg STX by caudal vein injection,the blank group was given the same amount of normal saline,and the poisoning symptoms were observed.The heart,liver,spleen,lung,kidney,stomach wall,brain,right lower limb muscle,blood and urine were dissected immediately after death and stored at-20℃ for examination.3.Data processingContent was represented by mean ± standard deviation,and data were processed and plotted using Graphpad Prism 8.3.0 software.Results:1.UPLC-MS/MS detection of saxitoxin and its metabolites:The results showed that in the blood samples,the linear relationship between saxitoxin,dicarbamoyl-saxitoxin and neosaxitoxin was good in the range of0.5～100ng/m L,1～100ng/m L and 1～100ng/m L,and the correlation coefficient(r)was ≥0.996.The limits of detection were 0.1ng/m L,0.5ng/m L and 0.5ng/m L,respectively,and the limits of quantitation were 0.5ng/m L,1ng/m L and 1ng/m L,respectively.The recoveries were 63.23%～81.77%,the intraday precision was 3.06%-9.58%,and the intraday precision was 3.87%～9.96%.In addition to urine samples,the three toxins showed a good linear relationship in the range of 1～100ng /m L,with correlation coefficient(r)≥ 0.996.The limit of detection was 0.5ng/m L,the limit of quantitation was1ng/m L,the recovery rate was 65.3%～74.34%,and the intra-day precision was3.18%～6.79%.Daytime precision was 5.60% ～7.81%.In the liver addition samples,the linear relationship between saxitoxin,dicarbamoyl-saxitoxin and neosaxitoxin was good in the range of 2～100ng /g,5～100ng /g and 5～100ng /g,respectively,with correlation coefficient(r)= 0.994.The limits of detection were 1ng/g,3ng/g and 2ng/g,respectively.The limits of quantitation were 2ng/g,5ng/g and 5ng/g,respectively.The recoveries were71.10%～112.01%,the intra-day precision was 1.69%～6.08%,and the intra-day precision was 4.07%～8.08%.2.Changes in blood concentration of saxitoxin in rats with acute poisoning:Oral intragastric administration: the low dose group of rats had no obvious toxic reaction,and no plasma and metabolites were detected in blood.High-dose rats showed lethargy,limb weakness,convulsion and other toxic reactions,and the blood saxitoxin reached the peak within 5min to 30 min,and then gradually decreased.the blood concentrations of dicarbamoyl-saxitoxin and neosaxitoxin showed an overall trend of increasing first and then decreasing,and the level of neosaxitoxin was higher than that of dicarbamoyl-saxitoxin.The difference in the death time of 108μg/kg rats suggested that the species and individuals of saxitoxin poisoning were different,and some saxitoxin and metabolites were detected in blood.Administered via tail vein: the toxic dose and death dose of rats were extremely close.The animals in the high-dose group died within 30 min,and the concentration of toxin in the heart blood at the time of death was positively correlated with the dose and the time of symptom occurrence.The concentration of saxitoxin in the blood of the low-dose group reached the peak within 1min～10min,and then began to decline.Neosaxitoxin could be detected within 10min～2.5h.The content of dicarbamoyl-saxitoxin in blood is low and only a few points can be detected.Part of the urine collected showed that the point at which the target was not detectable in the blood was detectable in the urine at a high level.3.Postmortem distribution of saxitoxin and its metabolites in poisoned rats:The concentration of saxitoxin in body fluids and organs of poisoned rats was in the order of liver,lung,spleen,blood,stomach wall > bladder > left kidney,right kidney >heart.The concentration of dicarbamoyl-saxitoxin in body fluids and organs of poisoned rats was in the order of stomach wall > bladder > liver,left kidney,right kidney > lung,heart,blood and spleen.The concentration of neosaxitoxin in body fluids and organs of poisoned rats was in the order of liver,left kidney,right kidney > spleen > heart,stomach wall,bladder > lung > heart blood.Conclusion:1.An UPLC-MS/MS method was established for the detection of saxitoxin,dicarbamoyl-saxitoxin and neosaxitoxin in biological samples.The method has strong specificity and sensitivity,and is suitable for the detection of toxins in blood,urine and liver samples in poisoning cases.2.In this study,the higher the dose,the more obvious the toxic reaction of rats,and the higher the concentration of toxin in the blood at the time of death.The contents of saxitoxin,dicarbamoyl-saxitoxin and neosaxitoxin in blood of rats reached the peak within a short time and then decreased,and the toxins were absorbed and eliminated quickly in the body.In rats,the poisoning window was narrow,the poisoning concentration was close to the lethal concentration,and the content of neosaxitoxin in vivo was higher than that of dicarbamoyl-saxitoxin,and the detection time was earlier,suggesting that neosaxitoxin may be the main metabolite of saxitoxin.3.Saxitoxin can be metabolized into dicarbamoyl-saxitoxin and neosaxitoxin in rats,which can be detected in most organs and body fluids of rats.The content of saxitoxin was higher in liver,lung,stomach wall,spleen and kidney,but lower in brain and muscle tissue.The content of dicarbamoyl-saxitoxin was low in all organs.Neosaxitoxin was mainly distributed in liver,kidney and spleen.In the case of death caused by saxitoxin poisoning,liver,kidney,lung and urine can be selected as materials for examination in addition to blood collection. |
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